Abstract:

Plants have been used in treatment and prevention of diseases for thousands of years. In
modern medicine there is a trend towards isolation and identification of bioactive molecules,
whereas plant preparations continue to be used in traditional medicinal systems. However,
combinations of drugs are usually administered in treatment of complex diseases like cancer
and HIV, in order to target multiple deregulated cellular pathways simultaneously.
Khat (Catha edulis Forsk.) has been cultivated for centuries primarily due to its use as a
natural stimulant. The stimulating potential of khat is mainly caused by its content of
cathinone, an alkaloid with structure and pharmacological profile similar to amphetamine.
Cathinone is a labile precursor for the less bioactive derivatives cathine and norephedrine.
An organic extract of khat was previously reported to induce programmed cell death in acute
myeloid leukemia (AML) cell lines, while being less toxic to normal peripheral blood
mononuclear cells (PBMCs). In this study, cellular and molecular effects of an extract of
khat were further elucidated in AML cell lines and compared with the cancer therapeutics
camptothecin. Early effects of khat and the khat amphetamines on intracellular signaling
responses in normal peripheral leukocytes were investigated, in addition to cytotoxic effects.
The khat extract was fractionated and analysed for cytotoxicity in AML cell lines, and the
fractions analysed by mass spectrometry.
The khat extract was shown to induce cell death in a subset of genetically diverse AML cell
lines, indicating involvement of specific mechanisms. In contrast to camptothecin, khat
caused structural damage to mitochondria and mediated impaired mitochondrial respiration.
In addition, khat was observed to induce formation of autophagosomes, indicating activation
of autophagy. The survival protein Bcl-2 protected against camptothecin, and partial
protection was obtained against khat-induced cell death. Procaspase-8 of the receptormediated
cell death pathway was activated by khat, while levels of the death antagonists
Mcl-1 and c-FLIPL were reduced. The stress sensor and tumour suppressor protein p53 was
induced and modulated in khat-mediated death, but not by sub-lethal dilutions of the khat
extract or in khat-resistant cells. Experiments using p53 knock-down and knock-out cells
demonstrated that khat-mediated cell death was independent of p53. The p53 protein was suggested to primarily act as a stress sensor in AML cells that were susceptible to khatinduced
cell death.
Fractionation of the khat extract and bio-guided screening in AML cell lines resulted in
identification of three separate cytotoxic fractions. The khat fractions were analysed by mass
spectrometry, which led to the partial characterization of a phenylpropanoid glycoside
suggested to represent the major cytotoxic constituent in the khat extract. In contrast to khat,
the khat amphetamines were observed to be relatively non-toxic to AML cell lines. Khat and
the khat amphetamines were shown to mediate early and generally opposite effects on
signaling mediators in normal immune cells. Whereas khat activated stress sensors, like p38
and p53, and demonstrated cytotoxic effects, the khat amphetamines attenuated activating
modifications of several signaling proteins, including p53, and appeared to have a
stimulating effect on lymphocyte proliferation.